Recently, a new class of fluorophore derivatives of tremendous value in the detection, visualization, and purification of recombinant proteins has been introduced.
These biarsenical fluorophore derivatives, as adducts with EDT (1,2-ethanedithiol), are self-quenching: the small size of the EDT moieties permits free rotation of the arsenic atoms, which quenches fluorescence of the fluorophore. The essentially nonfluorescent molecule is rendered fluorescent by competitive displacement of the EDT moiety by a specific tetracysteine peptide motif (CCXXCC, (SEQ ID NO: 1) where “X” represents any amino acid), an engineered sequence that is uncommon in natural proteins; binding to the tetracysteine motif constrains motion of the arsenic atoms, unquenching the fluorophore. Griffin et al., Science 281:269 (1998); Griffin et al., Methods Enzymol. 327:565-78 (2000); Adams et al., J. Amer. Chem. Soc. 124:6063-6076 (2002); Gaietta et al., Science 503-507 (2002); U.S. Pat. Nos. 5,932,474, 6,054,271; 6,451,569; 6,008,378; U.S. patent application publication no. 2003/0083373, and international patent application publication no. WO 99/21013, the disclosures of which are incorporated herein by reference in their entireties.
Advantages of the biarsenical fluorophores as fluorescent protein labeling reagents include small size, ability of the EDT2 adducts to cross cell membranes, ability to recognize a binding domain that is sufficiently small as not to interfere substantially with the function of the protein to which it is fused, nanomolar (or lower) dissociation constant for binding to the tetracysteine motif, rapid conversion from a nonfluorescent to a fluorescent state upon binding, and the reversibility of its binding upon addition of a high concentration (millimolar) of EDT.
The biarsenical derivative of fluorescein that is most commonly used is 4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2, known as FlAsH™-EDT2 or Lumio™ Green, and is available commercially (Invitrogen Corp., Carlsbad, Calif.). The red-fluorescing biarsenical resorufin derivative, known as ReAsH™ or Lumio™ Red, is also available commercially; methods of synthesizing other such biarsenical fluorophores, such as CHoXAsH-EDT2 and HoXAsH-EDT2 are described in the literature.
Tetracysteine biarsenical affinity tags (FlAsH™ tags) have been successfully incorporated at either the N- or C-termini of proteins, as well as exposed surface regions within a protein and have been used to permit visualization of recombinant proteins expressed within living cells, and in SDS-PAGE gels. Griffin et al. 1998, Griffin et al. 2000, Adams et al. 2002, supra.
In PAGE gels, inclusion of the FlAsH-EDT2 reagent in the sample loading buffer allows rapid detection of recombinant proteins in whole cell lysates using a standard UV light box without the need for western blotting or other more laborious protein detection methods.
Although the preferred tetracysteine motif occurs rarely in natural proteins, permitting specific labeling of proteins to which the tetracysteine motif has been recombinantly fused, FlAsH-EDT2 has been shown additionally to bind to endogenous cysteine-containing proteins, Stroffekova et al., Pflugers Arch.-Eur. J. Physiol. 442:859-866 (2001), which increases background fluorescence. Stroffekova et al. suggest that FlAsH™ binding to the vicinal cysteines in the C—X—X—C protein motif of endogenous proteins may limit the use of FlAsH-EDT2 to staining recombinant proteins expressed at a high level in cells with a naturally low background.
Given the advantages of biarsenical fluorophores as labeling and purification reagents for recombinantly tagged proteins, there is a need in the art for compositions, methods, and kits that permit tetracysteine-labeled fusion proteins to be labeled with biarsenical fluorophores with decreased reactivity with endogenous protein motifs. There is a particular need in the art for compositions, methods and kits that provide increased specificity of labeling of tetracysteine-tagged recombinant proteins resolved within electrophoresis gels.